It is proposed to characterize the proteins involved in guanine nucleotide regulation of hormone-sensitive adenylate chclase. Cholera toxin is used to alter the guanine nucleotide regulation of adenylate cyclase, and to identify specific regulatory proteins of the adenylate cyclase system. Cholera toxin, using (32P)NAD+, specifically rediolabels at lease two proteins in membranes of wild-type S49 lymphoma cells. A genetic variant of S49 cells (cyc minus) which is deficient in guanine nucleotide regalatory proteins also lacks the specific cholera toxin substrates. Differences in the number of specific cholera toxin substrates is apparent in cell types expressing different guane nucleotide regulatory properties suggesting that this may be related to the complexity of the hormone-sensitive adenylate cyclase system. The differences in functional properties of the regulatory proteins will be correlated with their differences in structure and number. Functional differences will be determined using three procedures: 1. Reconstitution assays using cyc minus membranes and detergent extracts from membranes of cell types having different adenylate cyclase regulatory properties. 2. Membrane-cell fusion techniques to build adenylate cyclase systems having specific regulatory proteins to examine their role in agonist-induced desensitization. 3. Heterokaryon and cell hybrid formation using avian erythrocytes and cyc minus cells to examine the re-expression of avian erythrocyte regulatory proteins. Structural differences will be determined by peptide mapping and isoelectric point determinations of the radiolabelled proteins that are specific substrates for cholera toxin. The regulatory proteins specifically labelled by cholera toxin will be partially purified, and after elution from an SDS-acrylamide gel used for the production of antisera against adenylate cyclase regulatory proteins.